This invention relates to polymerase chain reaction (PCR) amplification using microarrays of gel immobilized oligonucleotide primers in gel pads on a microchip (also known as a biochip). The invention also relates to a method of separating the individual gel pads on a microchip by immersing pads in a hydrophobic liquid, such as oil, as well as to novel structures for the gel pads which improves the efficacy of the PCR reactions in the immersed microchip. The primers can be selectively activated, inactivated, or detached from the gel matrix, and can be selected to perform combinatorial or multiplex PCR amplification.
DNA analysis using PCR amplification and detection of target regions of microorganisms is becoming increasingly popular for medical, environmental and military applications. For example, as the threat of international and domestic bioterrorism has heightened over the past few years, B. anthracis has arisen as a dangerous bioterrorist agent to be detected. Shiga and Shiga-like toxin genes of the toxin-producing strains of Shigella dysentariae and Escherichia coli, respectively, inhabiting human intestines are responsible for hemorrhagic colitis and other diseases. Diagnostic assays for these microorganism are useful. International and domestic epidemics of M. tuberculosis now include drug resistant strains, are growing in frequency, and now represent a serious public health problem facing the world. Rifampicin is one of the most efficient drugs used to treat tuberculosis patients, so detection is imperative, but mutations occurring within a short region of the rpoB gene account for rifampicin resistance in 96% of M. tuberculosis strains. Therefore, it is important to detect these mutations so appropriate treatment is used. Detection of a gene coding for xcex2-lactamase, which inactivates ampicillin, is a good model for screening for bacterial plasmid-born antibiotic resistance.
Polymerase Chain Reaction (PCR) amplification of specific DNA sequences is a powerful analytical tool that has many different applications, but which also has significant limitations. These limitations derive from the repetitious manual sample handling and analysis needed for many applications, i.e., the need for individual sample tubes and gel electrophoresis of the amplified end-products for analysis. At present, there is a rapidly growing need for parallel amplification and analysis of many different samples. Complex, sequential analysis is laborious.
PCR amplification is based on specific hybridization of oligonucleotide primers (relatively short pieces of DNA) that hybridize with longer DNA sequences of interest (target DNA). Currently PCR is usually performed in parallel experiments in different test tubes. Multiplex amplification, in which different amplification primers mixed with DNA molecules or DNA sample pools are carried through the PCR process, has a limit of less than ten different reactions in a single test tube. Therefore, a need exists to develop a simple and efficient method to amplify multiple DNA sequences with multiple primers, in a manner that allows the identification of specific amplified DNA fragments.
A desirable procedure would also be combining DNA amplification with detection and identification of specific sequences by hybridization. An easily used, cost effective method is also desirable.
The invention relates several general methods for performing PCR amplification combined with the detection and analysis of the PCR products on a microchip.
In one embodiment, PCR amplification takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid which separates the individual gel pads into environments which resemble micro-miniaturized test tubes. This method comprises the steps of: (a) providing a microchip with immobilized primers; (b) adding a hybridization solution containing a nucleic acid sequence to be tested to the microchip; (c) hybridizing the nucleic acid sequence to the primers; (d) replacing the hybridization solution with an amplification solution, which may also contain an enzyme or reagent which detaches/activates/inactivates a set of primers as discussed below; (e) removing the amplification solution and replacing it with a hydrophobic liquid, such as a hydrocarbon like mineral oil; (f) cycling the PCR at temperatures selected to be effective with one or more sets of PCR primers; and (g) repeating steps (a) to (f) for the different types of primers, thus performing a combinatorial PCR amplification on a microchip. One can use these methods of combinatorial amplification to design xe2x80x9cnestedxe2x80x9d primer experiments, to isolate and amplify sequences with increasing specificity with each repetition.
In another embodiment, the amplification occurs both outside in the solution surrounding the gel pads, and within a plurality of gel pads themselves on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. The method includes the steps of:
(a) providing a microchip with at least one oligonucleotide primer immobilized in gel pads;
(b) adding a solution to the microchip environment comprising an amplification buffer, primers and a nucleic acid sequence to be tested;
(c) cycling the temperature to achieve amplification; and
(d) repeating steps (a) to (c) while selectively activating, inactivating or detaching sets of primers within the gel pads.
The individual gel pads enclose a space capable of retaining the solution after the solution is removed from the microchip. At least one of the primers is immobilized in the gel pad. These immobilized primers can also be detachable primers, activatable primers, or inactivatable primers, selected depending on the needs of the experimenter.
For all embodiments there may be an additional step of running a primer extension or ligase reaction, or other related methods to detect and analyze the PCR products of the nucleic acid sequence to be tested.
Types of Primers
The composition of PCR primers suitable for practice of the invention are of two general types: 1) primers that are immobilized onto gel pads on a microchip and are active as PCR primers immediately after immobilization, and 2) primers that are modulated, which means that their activity as PCR primers can be changed by a separate treatment after immobilization.
There are at least three subtypes of modulated primers. Releasable or detachable (temporary immobilized) primers, are oligonucleotides that contain an oligoribonucleotide (RNA) linker incorporated into the 5xe2x80x2-terminal position of an oligodeoxynucleotide (DNA) primer that is immobilized onto the microchip gel pads, and that can subsequently be detached from the gel-pads by treatment with a ribonuclease. Upon treatment with a specific ribonuclease enzyme, the primer splits at the ribonucleotide residues and is released from the gel matrix within interior of the gel pad. Inactivatable (temporarily active) oligonucleotide primers, are immobilized oligonucleotides that can be inactivated. These primers contain, for example, an oligoribonucleotide chain in the middle or close to the 3xe2x80x2 terminal position, of an oligodeoxynucleotide primer. Upon treatment with a ribonuclease, these inactivatable primers can be inactivated.
Finally, activatable (temporarily inactive) oligonucleotide primers are immobilized primers that need to be activated in order to serve as primers. One example of an activatable primer has a phosphorylated 3xe2x80x2-terminal phosphate that prevents the primers from themselves being extended. To activate the primer, the phosphate group is removed by treatment with aphosphatase enzyme, thereby xe2x80x9cactivatingxe2x80x9d the primer. One suitable phosphatase enzyme is alkaline phosphatase.
The methods of the invention were applied for detection and mutation analysis of genes from a number of microorganisms of practical importance: anthrax toxin and shiga toxin genes, a plasmid-borne ampicillin resistance gene, and rifampicin-resistant genomic mutations of M. tuberculosis. 
On-chip amplification was applied to detect 1) anthrax toxin genes; and 2) a plasmid borne xcex2-lactamase gene responsible for bacterial ampicillin resistance. Allele specific on-chip amplification was used to identify a shiga toxin gene and discriminate it from a shiga-like toxin gene. Rifampicin resistant genomic mutations of Mycobacterium tuberculosis were detected by on-chip allele specific amplification of the rpoB region of DNA isolated from a human sputum. Gel pads with specific sets of mobilized primers may be separated from each other using mineral oil. Both amplification and hybridization are performed.
Definitions and Abbreviations
Gel matrix=composition such as polyacrylamide gel; gel pad=gel matrix units that form a microchip; ssDNA=single-stranded DNA; dsDNA=double-stranded DNA; F=forward primer; R=reverse primer; I=internal primers; *-P=hybridization oligonucleotide probe; PCR=polymerase chain reaction; and RNase=ribonuclease A.